The present invention relates to synthetic peptides derived from the primary sequence of the acute phase reactant C-reactive protein (CRP) (SEQ ID NO:1), more particularly to a peptide corresponding to positions 62-71 of CRP and derivatives thereof, which peptides inhibit in vitro the enzymatic activities of human leukocyte elastase (hLE) and/or human leukocyte cathepsin G (hCG), two potent serine proteases associated with tissue damage occurring in the course of several chronic inflammatory conditions. The invention further relates to anti-inflammatory pharmaceutical compositions comprising said CRP-derived peptides.
The following abbreviations will be used throughout the specification:
CRP, C-reactive protein; hLE, human leukocyte elastase; hCG, human leukocyte cathepsin G; MeOSuc-AAPV-NA, methoxysuccinyl-Ala-Ala-Pro-Val-nitroanilide (SEQ ID NO:2), Suc-AAPF-NA, succinyl-Ala-Ala-Pro-Phe-nitroanilide (SEQ ID NO:3).
C-reactive protein (CRP) is a plasma protein classified as a major acute phase reactant due to its dramatic accumulation in the blood stream during the inflammatory response. Within a relatively short period (24-48 hr) following tissue injury or certain traumatic events, the CRP blood concentration may rise 1000-fold over the normal level to as high as 1 mg/ml (Ballue and Kushner, 1992).
CRP consists of five identical sub-units that contain each 206 amino acids bridged by a single disulfide bond and that aggregate non-covalently into a cyclic pentamer termed pentraxin. The precise biochemical function of CRP as a whole entity is still obscure. CRP was shown to bind to specific receptors on human neutrophils (Kdxcx9c5xc3x9710xe2x88x928 M), monocytes (Kdxcx9c10xe2x88x927 M), and other inflammatory-related cells in vitro (Ballue and Kushner, 1992),
In the laboratories of the present inventors and their collaborators it was found that following binding to neutrophils, CRP is subsequently degraded by a membrane-associated neutral serine protease, which has been characterized (Shephard et al., 1992), and by lysosomal-derived enzymes to yield various low molecular weight peptides. Several of these peptides were identified, synthesized, and shown to be potent anti-inflammatory agents inhibiting neutrophil phagocytosis, degranulation, and superoxide ion (O2) generation (Shephard et al., 1990; Yavin et al., 1995). Superoxide ion is the parent compound of several destructive mediators that are believed to play a central role in inflammation-associated tissue injury (Ballue and Kushner, 1992).
The most prominent of the peptides disclosed by Shephard et al., 1990, and Yavin et al., 1995, were derived from within the primary sequence of CRP (SEQ ID NO:1) as follows: Asp70-Ile-Gly-Tyr-Ser74, Lys201-Pro-Gln-Leu-Trp-Pro206, Leu83-Phe-Glu-Val-Pro-Glu-Val-Thr90, Val-77-Gly-Gly-Ser-Glu-Ile82 (Shephard et al., 1990) and Asn160-Met-Trp-Asp-Phe-Val165, Gln203-Leu-Trp-Pro206, Ser18-Tyr-Val-Ser-Leu-Lys23 (Yavin et al., 1995). These peptides were shown by the authors to inhibit various neutrophilic functions, indicating that they may be capable of regulating superoxide ion production by neutrophils in vivo during the acute phase response as part of a complex protective mechanism. However, as disclosed in the PCT Publication No. WO 97/28182 of the same applicants, several of these peptides lack hLE inhibitory capability.
Human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG) are the two major potent neutral serine proteases found in the azurophilic granules of neutrophils which are involved in the intracellular digestion of proteins and play an important role in phagocytosis and host defense against invading organisms. In the extracellular environment, hLE is capable of degrading various connective tissue proteins including highly cross-linked elastin whereas hCG is very effective in degrading proteoglycans and collagens and has been shown to augment the elastolytic capability of hLE (Groutas, 1987).
The release of enzymes into the extracellular medium by activated neutrophils is normally controlled by several potent inhibitors. The most specific natural inhibitors, xcex11-protease inhibitor (xcex11-PI) and xcex1-antichymotrypsin (ACT), are directed against hLE and hCG, respectively (Groutas, 1987). Imbalances in the levels of tissue proteases such as hLE and hCG, and their inhibitors, allow excess hLE and hCG to attack connective tissue, and are implicated in the severe and permanent tissue damage associated with pulmonary emphysema (Groutas, 1987), rheumatoid arthritis (Gallin et al., 1988), cystic fibrosis (Jackson et al., 1984) and several other inflammatory conditions. Major research efforts have been dedicated to develop potent inhibitors of hLE and hCG based on a wide variety of low molecular weight organic compounds (Edwards and Bernstein, 1994) such as 3,3-dialkylazetidin-2-ones, proposed as orally active xcex2-lactam inhibitors of hLE (Finke et al., 1995).
CRP as a whole protein was reported to have no inhibitory effect on hLE (Vachino et al., 1988). In contrast, a specific region within the primary sequence of CRP containing the core peptide Val89-Thr-Val-Ala-Pro-Val-His-Ile96 of SEQ ID NO:1 was shown to inhibit in vitro the enzymatic activities of hLE and hCG to a larger extent than peptides of similar chain lengths corresponding to the active sites of their natural inhibitors (PCT Publication No. WO 97/28182; Yavin et al., 1996). Novel biologically active CRP-derived peptides, i.e. peptides capable of inhibiting in vitro the enzymatic activity of hLE and/or of hCG, previously concealed within the inner hydrophobic disulfide loop which spans CRP36-97 in each subunit (see FIG. 1), have been found in accordance with the present invention to significantly inhibit the enzymatic activities of hLE and hCG enzymes.
The present invention relates to a synthetic CRP-derived peptide capable of inhibiting in vitro the enzymatic activity of human leukocyte elastase (hLE) and/or of human cathepsin G (hCG), said peptide being selected from:
(i) a core peptide corresponding to positions 62-71 of the sequence of human C-reactive protein (CRP) of the formula:
Glu62-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile71 
or peptides resulting from modification thereof characterized by:
(ii) substitution of Glu62 by Asp or by a non-amino acid negatively charged residue derived from succinic, glutaric or adipic acids;
(iii) substitution of each of the residues Ile63, Leu64, Ile65, Phe66 or Trp67 by a natural or non-natural hydrophobic amino acid residue;
(iv) deletion of 1 or 2 amino acid residues selected from Ile63, Leu64, Ile65, Phe66 and Trp67;
(v) substitution of 1-3 amino acid residues selected from Ile63, Leu64, Ile65, Phe66 and Trp67 by a single non-natural amino acid residue derived from 6-aminocaproic acid;
(vi) substitution of 2-4 amino acid residues selected from Ile63, Leu64, Ile65, Phe66 and Trp67 by a single non-natural amino acid residue derived from 8-aminocaproic acid, 10-aminodecanoic acid or 12-aminolauric acid;
(vii) substitution of 3-5 amino acid residues selected from Ile63, Leu64, Ile65, Phe66 and Trp67 by a stretch of identical hydrophobic amino acid residues or by a single non-natural amino acid residue derived from 10-aminodecanoic acid or 12-aminolauric acid;
(viii) substitution of Ser68 by a natural or non-natural amino acid residue selected from Thr, Cys, Ala and homoserine;
(ix) substitution of Lys69 by a natural or non-natural positively charged or hydrophobic amino acid residue;
(x) substitution of Asp70 by a negatively charged or a polar amino acid residue selected from Glu, Asn or Gln;
(xi) substitution of Ile71 by a natural or non-natural hydrophobic amino acid residue;
(xii) elongation of a peptide (i) to (xi) by 1-5 non-charged amino acid residues at the N-terminus and/or at the C-terminus;
(xiii) substitution of any amino acid residue in a peptide (i) to (xii) by the corresponding N-alkyl derivative, D-amino acid residue or by another isoster;
(xiv) an amide of the C-terminal of a peptide (i) to (xiii); and
(xv) an N-acyl derivative of a peptide (i) to (xiv).
The invention further relates to anti-inflammatory pharmaceutical compositions comprising a CRP-derived peptide of the invention and a pharmaceutically acceptable carrier.
In another aspect, the invention relates to a method of treatment of an inflammatory disorder, e.g. rheumatoid arthritis, pulmonary emphysema, cystic fibrosis, bronchitis, asthma, acute respiratory distress syndrome and other chronic inflammatory tissue destructive conditions, which comprises administering to a patient in need thereof an effective amount of a CRP-derived peptide according to the invention.